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Prostate cancer (PCa), the second leading cause of death in American men, includes distinct genetic subtypes with distinct therapeutic vulnerabilities. The DACH1gene encodes a winged helix/Forkhead DNA-binding protein that competes for binding to FOXM1 sites. Herein, DACH1gene deletion within the 13q21.31-q21.33 region occurs in up to 18% of human PCa and was associated with increased AR activity and poor prognosis. In prostate OncoMice, prostate-specific deletion of the Dach1gene enhanced prostatic intraepithelial neoplasia (PIN), and was associated with increased TGFβ activity and DNA damage. Reduced Dach1increased DNA damage in response to genotoxic stresses. DACH1was recruited to sites of DNA damage, augmenting recruitment of Ku70/Ku80. Reduced Dach1expression was associated with increased homology directed repair and resistance to PARP inhibitors and TGFβ kinase inhibitors. Reduced Dach1expression may define a subclass of PCa that warrants specific therapies.

As severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to circulate, multiple variants of concern have emerged. New variants pose challenges for diagnostic platforms because sequence diversity can alter primer/probe-binding sites (PBSs), causing false-negative results. The MassARRAY SARS-CoV-2 Panel (Agena Bioscience) uses RT-PCR and mass spectrometry to detect five multiplex targets across Nand ORF1abgenes. Herein, we use a data set of 256 SARS-CoV-2–positive specimens collected between April 11, 2021, and August 28, 2021, to evaluate target performance with paired sequencing data. During this time frame, two targets in the Ngene (N2 and N3) were subject to the greatest sequence diversity. In specimens with N3 dropout, 69% harbored the Alpha-specific A28095U polymorphism that introduces a 3′-mismatch to the N3 forward PBS and increases risk of target dropout relative to specimens with 28095A (relative risk, 20.02; 95% CI, 11.36 to 35.72; P < 0.0001). Furthermore, among specimens with N2 dropout, 90% harbored the Delta-specific G28916U polymorphism that creates a 3′-mismatch to the N2 probe PBS and increases target dropout risk (relative risk, 11.92; 95% CI, 8.17 to 14.06; P < 0.0001). These findings highlight the robust capability of MassARRAY SARS-CoV-2 Panel target results to reveal circulating virus diversity, and they underscore the power of multitarget design to capture variants of concern.

Current understanding of coronavirus disease 2019 (COVID-19) pathophysiology is limited by disease heterogeneity, complexity, and a paucity of studies assessing patient tissues with advanced molecular tools. Rapid autopsy tissues were evaluated using multiscale, next-generation RNA-sequencing methods (bulk, single-nuclei, and spatial transcriptomics) to provide unprecedented molecular resolution of COVID-19-induced damage. Comparison of infected/uninfected tissues revealed four major regulatory pathways. Effectors within these pathways could constitute novel therapeutic targets, including the complement receptor C3AR1, calcitonin receptor–like receptor, or decorin. Single-nuclei RNA sequencing of olfactory bulb and prefrontal cortex highlighted remarkable diversity of coronavirus receptors. Angiotensin-converting enzyme 2 was rarely expressed, whereas basigin showed diffuse expression, and alanyl aminopeptidase, membrane, was associated with vascular/mesenchymal cell types. Comparison of lung and lymph node tissues from patients with different symptoms (one had died after a month-long hospitalization with multiorgan involvement, and the other had died after a few days of respiratory symptoms) with digital spatial profiling resulted in distinct molecular phenotypes. Evaluation of COVID-19 rapid autopsy tissues with advanced molecular techniques can identify pathways and effectors, map diverse receptors at the single-cell level, and help dissect differences driving diverging clinical courses among individual patients. Extension of this approach to larger data sets will substantially advance the understanding of the mechanisms behind COVID-19 pathophysiology.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated clinical syndrome COVID-19 are causing overwhelming morbidity and mortality around the globe and disproportionately affected New York City between March and May 2020. Here, we report on the first 100 COVID-19-positive autopsies performed at the Mount Sinai Hospital in New York City. Autopsies revealed large pulmonary emboli in six cases. Diffuse alveolar damage was present in over 90% of cases. We also report microthrombi in multiple organ systems including the brain, as well as hemophagocytosis. We additionally provide electron microscopic evidence of the presence of the virus in our samples. Laboratory results of our COVID-19 cohort disclose elevated inflammatory markers, abnormal coagulation values, and elevated cytokines IL-6, IL-8, and TNFα. Our autopsy series of COVID-19-positive patients reveals that this disease, often conceptualized as a primarily respiratory viral illness, has widespread effects in the body including hypercoagulability, a hyperinflammatory state, and endothelial dysfunction. Targeting of these multisystemic pathways could lead to new treatment avenues as well as combination therapies against SARS-CoV-2 infection.

Prostate cancer is diagnosed mostly in men over the age of 50 years, and has favorable 5-year survival rates due to early cancer detection and availability of curative surgical management. However, progression to metastasis and emergence of therapeutic resistance are responsible for the majority of prostate cancer mortalities. Recent advancement in sequencing technologies and computational capabilities have improved the ability to organize and analyze large data, thus enabling the identification of novel biomarkers for survival, metastatic progression and patient prognosis. Large-scale sequencing studies have also uncovered genetic and epigenetic signatures associated with prostate cancer molecular subtypes, supporting the development of personalized targeted-therapies. However, the current state of mainstream prostate cancer management does not take full advantage of the personalized diagnostic and treatment modalities available. This review focuses on interrogating biomarkers of prostate cancer progression, including gene signatures that correspond to the acquisition of tumor lethality and those of predictive and prognostic value in progression to advanced disease, and suggest how we can use our knowledge of biomarkers and molecular subtypes to improve patient treatment and survival outcomes.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a new human disease with few effective treatments1. Convalescent plasma, donated by persons who have recovered from COVID-19, is the acellular component of blood that contains antibodies, including those that specifically recognize SARS-CoV-2. These antibodies, when transfused into patients infected with SARS-CoV-2, are thought to exert an antiviral effect, suppressing virus replication before patients have mounted their own humoral immune responses2,3. Virus-specific antibodies from recovered persons are often the first available therapy for an emerging infectious disease, a stopgap treatment while new antivirals and vaccines are being developed1,2. This retrospective, propensity score–matched case–control study assessed the effectiveness of convalescent plasma therapy in 39 patients with severe or life-threatening COVID-19 at The Mount Sinai Hospital in New York City. Oxygen requirements on day 14 after transfusion worsened in 17.9% of plasma recipients versus 28.2% of propensity score–matched controls who were hospitalized with COVID-19 (adjusted odds ratio (OR), 0.86; 95% confidence interval (CI), 0.75–0.98; chi-square test Pvalue?=?0.025). Survival also improved in plasma recipients (adjusted hazard ratio (HR), 0.34; 95% CI, 0.13–0.89; chi-square test P?=?0.027). Convalescent plasma is potentially effective against COVID-19, but adequately powered, randomized controlled trials are needed.

Several studies have revealed that the hyper-inflammatory response induced by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a major cause of disease severity and death. However, predictive biomarkers of pathogenic inflammation to help guide targetable immune pathways are critically lacking. We implemented a rapid multiplex cytokine assay to measure serum interleukin (IL)-6, IL-8, tumor necrosis factor (TNF)-α and IL-1β in hospitalized patients with coronavirus disease 2019 (COVID-19) upon admission to the Mount Sinai Health System in New York. Patients (n= 1,484) were followed up to 41 d after admission (median, 8 d), and clinical information, laboratory test results and patient outcomes were collected. We found that high serum IL-6, IL-8 and TNF-α levels at the time of hospitalization were strong and independent predictors of patient survival (P< 0.0001, P= 0.0205 and P= 0.0140, respectively). Notably, when adjusting for disease severity, common laboratory inflammation markers, hypoxia and other vitals, demographics, and a range of comorbidities, IL-6 and TNF-α serum levels remained independent and significant predictors of disease severity and death. These findings were validated in a second cohort of patients (n= 231). We propose that serum IL-6 and TNF-α levels should be considered in the management and treatment of patients with COVID-19 to stratify prospective clinical trials, guide resource allocation and inform therapeutic options.

Prostatic intraepithelial neoplasia is a precursor to prostate cancer. Herein, deletion of the NAD+-dependent histone deacetylase SIRT1induced histological features of prostatic intraepithelial neoplasia at 7 months of age, which were associated with increased cell proliferation and enhanced mitophagy. In human prostate cancer, lower SIRT1expression in the luminal epithelium was associated with poor prognosis. Genetic deletion of SIRT1increased mitochondrial superoxide dismutase 2 acetylation of lysine residue 68, thereby enhancing reactive oxygen species production and reducing superoxide dismutase 2 activity. The PARK2gene, which has several features of a tumor suppressor, encodes an E3 ubiquitin ligase that participates in removal of damaged mitochondria via mitophagy. Increased reactive oxygen species in SIRT1-/-cells enhanced the recruitment of PARK2 to the mitochondria, inducing mitophagy. SIRT1restoration inhibited PARK2 translocation and reactive oxygen species production requiring the SIRT1 catalytic domain. Thus, the NAD+-dependent inhibition of superoxide dismutase 2 activity and reactive oxygen species by SIRT1 provides a gate keeper function to reduce PARK2-mediated mitophagy and aberrant cell survival.

circRNAs arise from back splicing events during mRNA processing, and when deregulated can play an active role in cancer. Here we characterize a new circRNA (circPOK) encoded by the Zbtb7agene (also kown as POKEMON, LRF) in the context of mesenchymal tumor progression. circPOK functions as a non-coding proto-oncogenic RNA independently and antithetically to its linear transcript counterpart, which acts as a tumor suppressor by encoding the Pokemon transcription factor. We find that circPOK regulates pro-proliferative and pro-angiogenic factors by co-activation of the ILF2/3 complex. Importantly, the expression of Pokemon protein and circRNA is aberrantly uncoupled in cancer through differential post-transcriptional regulation. Thus, we identify a novel type of genetic unit, the iRegulon, that yields biochemically distinct RNA products, circular and linear, with diverse and antithetical functions. Our findings further expand the cellular repertoire towards the control of normal biological outputs, while aberrant expression of such components may underlie disease pathogenesis including cancer.

Accumulation of abnormal tau in neurofibrillary tangles (NFT) occurs in Alzheimer disease (AD) and a spectrum of tauopathies. These tauopathies have diverse and overlapping morphological phenotypes that obscure classification and quantitative assessments. Recently, powerful machine learning-based approaches have emerged, allowing the recognition and quantification of pathological changes from digital images. Here, we applied deep learning to the neuropathological assessment of NFT in postmortem human brain tissue to develop a classifier capable of recognizing and quantifying tau burden. The histopathological material was derived from 22 autopsy brains from patients with tauopathies. We used a custom web-based informatics platform integrated with an in-house information management system to manage whole slide images (WSI) and human expert annotations as ground truth. We utilized fully annotated regions to train a deep learning fully convolutional neural network (FCN) implemented in PyTorch against the human expert annotations. We found that the deep learning framework is capable of identifying and quantifying NFT with a range of staining intensities and diverse morphologies. With our FCN model, we achieved high precision and recall in naive WSI semantic segmentation, correctly identifying tangle objects using a SegNet model trained for 200 epochs. Our FCN is efficient and well suited for the practical application of WSIs with average processing times of 45 min per WSI per GPU, enabling reliable and reproducible large-scale detection of tangles. We measured performance on test data of 50 pre-annotated regions on eight naive WSI across various tauopathies, resulting in the recall, precision, and an F1 score of 0.92, 0.72, and 0.81, respectively. Machine learning is a useful tool for complex pathological assessment of AD and other tauopathies. Using deep learning classifiers, we have the potential to integrate cell- and region-specific annotations with clinical, genetic, and molecular data, providing unbiased data for clinicopathological correlations that will enhance our knowledge of the neurodegeneration.

Accumulation of abnormal tau in neurofibrillary tangles (NFT) occurs in Alzheimer disease (AD) and a spectrum of tauopathies. These tauopathies have diverse and overlapping morphological phenotypes that obscure classification and quantitative assessments. Recently, powerful machine learning-based approaches have emerged, allowing the recognition and quantification of pathological changes from digital images. Here, we applied deep learning to the neuropathological assessment of NFT in postmortem human brain tissue to develop a classifier capable of recognizing and quantifying tau burden. The histopathological material was derived from 22 autopsy brains from patients with tauopathies. We used a custom web-based informatics platform integrated with an in-house information management system to manage whole slide images (WSI) and human expert annotations as ground truth. We utilized fully annotated regions to train a deep learning fully convolutional neural network (FCN) implemented in PyTorch against the human expert annotations. We found that the deep learning framework is capable of identifying and quantifying NFT with a range of staining intensities and diverse morphologies. With our FCN model, we achieved high precision and recall in naive WSI semantic segmentation, correctly identifying tangle objects using a SegNet model trained for 200 epochs. Our FCN is efficient and well suited for the practical application of WSIs with average processing times of 45 min per WSI per GPU, enabling reliable and reproducible large-scale detection of tangles. We measured performance on test data of 50 pre-annotated regions on eight naive WSI across various tauopathies, resulting in the recall, precision, and an F1 score of 0.92, 0.72, and 0.81, respectively. Machine learning is a useful tool for complex pathological assessment of AD and other tauopathies. Using deep learning classifiers, we have the potential to integrate cell- and region-specific annotations with clinical, genetic, and molecular data, providing unbiased data for clinicopathological correlations that will enhance our knowledge of the neurodegeneration.

Whereas COVID-19 mRNA vaccines have shown remarkable efficacy in the prevention of severe disease and mortality in healthy individuals, the effectiveness in cancer patients has been more variable. Patients with multiple myeloma (MM) are at a high risk for severe infection due to disease- or treatment-related immune suppression. Our prior work demonstrated that the immune response to SARS-CoV-2 immunization in MM patients with 2 doses of mRNA vaccine was suboptimal and that a 3rd dose significantly improved neutralization of viral variants. MM patients undergoing treatment targeting BCMA were among those at a higher risk of ineffective immune responses. As BCMA-targeted (CAR T) treatment becomes more widely available, guidance on initiation of vaccination after treatment is needed.

Background:In response to the ever-evolving challenges posed by the antigenically diverse Omicron variants, the deployment of bivalent mRNA booster vaccines encompassing both the ancestral (WA1) and Omicron BA.5 spike components was initiated in fall of 2022. The real-world impact of these bivalent vaccines is largely unknown in patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster implementing cutting edge methodologies to identify patterns associated with continuing vulnerability in the current post-pandemic era.

Bladder cancers can be categorized into subtypes according to gene expression patterns. P53-like muscle-invasive bladder cancers are generally resistant to cisplatin-based chemotherapy, but exhibit heterogeneous clinical outcomes with a prognosis intermediate to that of the luminal and basal subtypes. The optimal approach to p53-like tumors remains poorly defined and better means to risk-stratify such tumors and identification of novel therapeutic targets is urgently needed. MicroRNAs (miRNAs) play a key role in cancer, both in tumorigenesis and tumor progression. In the past few years, miRNA expression signatures have been reported as prognostic biomarkers in different tumor types including bladder cancer. However, miRNA’s expression does not always correlate well with its activity. We previously developed ActMiR, a computational method for explicitly inferring miRNA activities. We applied ActMiRto The Cancer Genome Atlas (TCGA) bladder cancer data set and identified the activities of miR-106b-5p and miR-532-3p as potential prognostic markers of the p53-like subtype, and validated them in three independent bladder cancer data sets. Especially, higher miR-106b-5p activity was consistently associated with better survival in these data sets. Furthermore, we experimentally validated causal relationships between miR-106-5p and its predicted target genes in p53-like cell line HT1197. HT1197 cells treated with the miR-106b-5p-specific inhibitor were more invasive while cells treated with the miR-106b-5p-specific mimic were less invasive than corresponding controls. Altogether, our results suggest that miR-106b-5p activity can categorize p53-like bladder tumors into more and less-favorable prognostic groups, which provides critical information for personalizing treatment option for p53-like bladder cancers.

Postoperative risk assessment remains an important variable in the effective treatment of prostate cancer. There is an unmet clinical need for a test with the potential to enhance the Gleason grading system with novel features that more accurately reflect a personalized prediction of clinical failure. A prospectively designed retrospective study utilizing 892 patients, post radical prostatectomy, followed for a median of 8 years. In training, using digital image analysis to combine microscopic pattern analysis/machine learning with biomarkers, we evaluated Precise Post-op model results to predict clinical failure in 446 patients. The derived prognostic score was validated in 446 patients. Eligible subjects required complete clinical-pathologic variables and were excluded if they had received neoadjuvant treatment including androgen deprivation, radiation or chemotherapy prior to surgery. No patients were enrolled with metastatic disease prior to surgery. Evaluate the assay using time to event concordance index (C-index), Kaplan–Meier, and hazards ratio. In the training cohort (n= 306), the Precise Post-op test predicted significant clinical failure with a C-index of 0.82, [95% CI: 0.76–0.86], HR:6.7, [95% CI: 3.59–12.45], p< 0.00001. Results were confirmed in validation (n= 284) with a C-index 0.77 [95% CI: 0.72–0.81], HR = 5.4, [95% CI: 2.74–10.52], p< 0.00001. By comparison, a clinical feature base model had a C-index of 0.70 with a HR = 3.7. The Post-Op test also re-classified 58% of CAPRA-S intermediate risk patients as low risk for clinical failure. Precise Post-op tissue-based test discriminates low from intermediate high risk prostate cancer disease progression in the postoperative setting. Guided by machine learning, the test enhances traditional Gleason grading with novel features that accurately reflect the biology of personalized risk assignment.

Lipids, either endogenously synthesized or exogenous, have been linked to human cancer. Here we found that PMLis frequently co-deleted with PTENin metastatic human prostate cancer (CaP). We demonstrated that conditional inactivation of Pmlin the mouse prostate morphs indolent Pten-null tumors into lethal metastatic disease. We identified MAPK reactivation, subsequent hyperactivation of an aberrant SREBP prometastatic lipogenic program, and a distinctive lipidomic profile as key characteristic features of metastatic Pmland Ptendouble-null CaP. Furthermore, targeting SREBP in vivo by fatostatin blocked both tumor growth and distant metastasis. Importantly, a high-fat diet (HFD) induced lipid accumulation in prostate tumors and was sufficient to drive metastasis in a nonmetastatic Pten-null mouse model of CaP, and an SREBP signature was highly enriched in metastatic human CaP. Thus, our findings uncover a prometastatic lipogenic program and lend direct genetic and experimental support to the notion that a Western HFD can promote metastasis.

Activation of the PTEN–PI3K–mTORC1 pathway consolidates metabolic programs that sustain cancer cell growth and proliferation. Here we show that mechanistic target of rapamycin complex 1 (mTORC1) regulates polyamine dynamics, a metabolic route that is essential for oncogenicity. By using integrative metabolomics in a mouse model and human biopsies of prostate cancer, we identify alterations in tumours affecting the production of decarboxylated S-adenosylmethionine (dcSAM) and polyamine synthesis. Mechanistically, this metabolic rewiring stems from mTORC1-dependent regulation of S-adenosylmethionine decarboxylase 1 (AMD1) stability. This novel molecular regulation is validated in mouse and human cancer specimens. AMD1 is upregulated in human prostate cancer with activated mTORC1. Conversely, samples from a clinical trial with the mTORC1 inhibitor everolimus exhibit a predominant decrease in AMD1 immunoreactivity that is associated with a decrease in proliferation, in line with the requirement of dcSAM production for oncogenicity. These findings provide fundamental information about the complex regulatory landscape controlled by mTORC1 to integrate and translate growth signals into an oncogenic metabolic program.

PTEN, a known tumour suppressor, inhibits the FXBL2-dependent degradation of IP3R3, an IP3 receptor, thus augmenting IP3R3-mediated calcium release from the endoplasmic reticulum to mitochondria and inducing apoptosis; inhibiting FXBL2 sensitizes PTEN-deficient tumours to photodynamic therapy.

Advanced prostate cancer is a classic example of the intractability and consequent lethality that characterizes metastatic carcinomas. Novel treatments have improved the survival of men with prostate cancer; however, advanced prostate cancer invariably becomes resistant to these therapies and ultimately progresses to a lethal metastatic stage. Consequently, detailed knowledge of the molecular mechanisms that control prostate cancer cell survival and progression towards this lethal stage of disease will benefit the development of new therapeutics. The transcription factor endothelial transcription factor GATA-2 (GATA2) has been reported to have a key role in driving prostate cancer aggressiveness. In addition to being a pioneer transcription factor that increases androgen receptor (AR) binding and activity, GATA2 regulates a core subset of clinically relevant genes in an AR-independent manner. Functionally, GATA2 overexpression in prostate cancer increases cellular motility and invasiveness, proliferation, tumorigenicity, and resistance to standard therapies. Thus, GATA2 has a multifaceted function in prostate cancer aggressiveness and is a highly attractive target in the development of novel treatments against lethal prostate cancer.

Increased polyamine synthesis is known to play an important role in prostate cancer. We aimed to explore its functional significance in prostate tumor initiation and its link to androgen receptor (AR) signaling. For this purpose, we generated a new cell line derived from normal epithelial prostate cells (RWPE-1) with overexpression of ornithine decarboxylase (ODC) and used it for in vitroand in vivoexperiments. We then comprehensively analyzed the expression of the main metabolic enzymes of the polyamine pathway and spermine abundance in 120 well-characterized cases of human prostate cancer and high-grade prostate intraepithelial neoplasia (HGPIN). Herein, we show that the ODC-overexpressing prostate cells underwent malignant transformation, revealing that ODC is sufficient for de novo tumor initiation in 94% of injected mice. This oncogenic capacity was acquired through alteration of critical signaling networks, including AR, EIF2, and mTOR/MAPK. RNA silencing experiments revealed the link between AR signaling and polyamine metabolism. Human prostate cancers consistently demonstrated up-regulation of the main polyamine enzymes analyzed (ODC, polyamine oxidase, and spermine synthase) and reduction of spermine. This phenotype was also dominant in HGPIN, rendering it a new biomarker of malignant transformation. In summary, we report that ODC plays a key role in prostate tumorigenesis and that the polyamine pathway is altered as early as HGPIN.

Metastatic prostate cancer (PCa) inevitably acquires resistance to standard therapy preceding lethality. Here, we unveil a chromosomal instability (CIN) tolerance mechanism as a therapeutic vulnerability of therapy-refractory lethal PCa. Through genomic and transcriptomic analysis of patient datasets, we find that castration and chemotherapy-resistant tumors display the highest CIN and mitotic kinase levels. Functional genomics screening coupled with quantitative phosphoproteomics identify MASTL kinase as a survival vulnerability specific of chemotherapy-resistant PCa cells. Mechanistically, MASTL upregulation is driven by transcriptional rewiring mechanisms involving the non-canonical transcription factors androgen receptor splice variant 7 and E2F7 in a circuitry that restrains deleterious CIN and prevents cell death selectively in metastatic therapy-resistant PCa cells. Notably, MASTL pharmacological inhibition re-sensitizes tumors to standard therapy and improves survival of pre-clinical models. These results uncover a targetable mechanism promoting high CIN adaptation and survival of lethal PCa.

Leishmaniasis is a complex vector-borne disease caused by various Leishmaniaspecies, affecting humans and animals. Current diagnostic methods have limitations, leading to potential misdiagnosis. Therefore, there is an urgent need for specific and sensitive diagnostic tools. We evaluated the sensitivity of a quantitative real-time PCR (qPCR) assay targeting the 18S gene in diverse clinical sample matrices. The assay showed a wide dynamic range and a limit of detection (LoD) of 1 parasite equivalent per milliliter (eq-p/mL) for all tested species. It exhibited high specificity for LeishmaniaDNA, with no amplification against other microorganisms. When applied to samples from patients with visceral and cutaneous leishmaniasis, the qPCR assay provided results that matched the reference methods and allowed estimation of parasite burdens. This assay holds promise for diagnosing and monitoring leishmaniasis by offering high sensitivity, specificity, and the ability to estimate parasitemia. Further studies are needed to enhance Leishmaniamolecular diagnostics and expand their coverage for improved clinical impact.

Cellular transformation and cancer progression is accompanied by changes in the metabolic landscape. Master co-regulators of metabolism orchestrate the modulation of multiple metabolic pathways through transcriptional programs, and hence constitute a probabilistically parsimonious mechanism for general metabolic rewiring. Here we show that the transcriptional co-activator peroxisome proliferator-activated receptor gamma co-activator 1α (PGC1α) suppresses prostate cancer progression and metastasis. A metabolic co-regulator data mining analysis unveiled that PGC1α is downregulated in prostate cancer and associated with disease progression. Using genetically engineered mouse models and xenografts, we demonstrated that PGC1α opposes prostate cancer progression and metastasis. Mechanistically, the use of integrative metabolomics and transcriptomics revealed that PGC1α activates an oestrogen-related receptor alpha (ERRα)-dependent transcriptional program to elicit a catabolic state and metastasis suppression. Importantly, a signature based on the PGC1α–ERRα pathway exhibited prognostic potential in prostate cancer, thus uncovering the relevance of monitoring and manipulating this pathway for prostate cancer stratification and treatment.

For patients newly diagnosed with prostate cancer, the most significant question is whether the ‘truly malignant’ disease has been identified. This review will provide an overview of current prostate cancer genomic and biomarker discovery – validation strategies geared towards identifying aggressive, clinically significant disease at the time of diagnosis.

Soft tissue sarcomas are a heterogeneous group of tumors associated with poor clinical outcome. Although a subset of soft tissue sarcomas is characterized by simple karyotypes and recurrent chromosomal translocations, the mechanisms driving cytogenetically complex sarcomas are largely unknown. Clinical evidence led us to partially inactivate Ptenand Tp53in the smooth muscle lineage of mice, which developed high-grade undifferentiated pleomorphic sarcomas, leiomyosarcomas, and carcinosarcomas that widely recapitulate the human disease, including the aberrant karyotype and metastatic behavior. Ptenwas found haploinsufficient, whereas the wild-type allele of Tp53invariably gained point mutations. Gene expression profiles showed up-regulated Notch signaling in PtenΔ/+Tp53Δ/+tumors compared with Pten+/+Tp53Δ/+tumors. Consistently, Ptensilencing exacerbated the clonogenic and invasive potential of Tp53-deficient bone marrow–derived mouse mesenchymal stem cells and tumor cells and activated the Notch pathway. Moreover, the increased oncogenic behavior of PtenΔ/+Tp53Δ/+and shPten-transduced Pten+/+Tp53Δ/+tumor cells was counteracted by treatment with a γ-secretase inhibitor, suggesting that the aggressiveness of those tumors can be attributed, at least in part, to enhanced Notch signaling. This study demonstrates a cooperative role for Ptenand Tp53suppression in complex karyotype sarcomas while establishing Notch as an important functional player in the cross talk of these pathways during tumor progression. Our results highlight the importance of molecularly subclassifying patients with high-grade sarcoma for targeted treatments.

For men newly diagnosed with prostate cancer, there are limited tools to understand the risk of disease progression and guide the treatment decision process. We will provide an overview of current prostate cancer biomarker discovery and validation strategies that are geared toward identifying aggressive, clinically significant disease at the time of diagnosis.

miRNAs are small noncoding RNAs with critical roles in a large variety of biological processes such as development and tumorigenesis. miRNA expression profiling has been reported to be a powerful tool to classify tissue samples, including cancers, based on their developmental lineage. In this study, we have profiled the expression of miRNAs in bladder carcinoma in situ(CIS) and distinct cell compartments of the normal bladder, namely umbrella and basal-intermediate urothelial cells, as well as the muscularis propria. We identified several miRNAs differentially expressed between umbrella and basal-intermediate cells (miR-133a, miR-139-3p, miR-142-3p, miR-199b-5p, and miR-221). In situhybridization confirmed the expression of miR-133a and miR-139-3p in umbrella cells, and miR-142-3p in basal-intermediate cells. Strikingly, miRNA expression levels of CIS most closely resembled the miRNA profile of umbrella cells. Finally, we examined well-established umbrella and basal-intermediate cell immunohistochemical biomarkers in an independent series of CIS samples. Again, this analysis revealed the significant expression of umbrella-specific markers in CIS when compared to non-CIS lesions. Overall, our studies represent a comprehensive and accurate description of the different miRNAs expressed in CIS tumors and three distinct histological areas of the urinary bladder. Notably, this study provides evidence of the possible origin relationship between CIS and normal umbrella cells.

The promyelocytic leukemia (PML) tumor suppressor gene was initially identified as part of the t(15:17) chromosomal translocation associated with acute promyelocytic leukemia (APL). The PML protein is responsible for the assembly and function of characteristic nuclear domains known as PML-nuclear bodies (PML-NBs), which have been implicated in a variety of cellular functions, including growth suppression, apoptosis, and cellular senescence. PML’s many roles have been linked, at least in part, to its functional interaction with the tumor suppressor p53. It has been shown that PML favors both p53 accumulation and transcriptional activity; in turn, PML expression is directly regulated by p53, and this reciprocal regulation contributes to p53-mediated apoptosis and senescence. Nevertheless, genetic proof and in vivoassessment of the relevance of this functional crosstalk are still missing. Here we show that complete Pmlinactivation, in a context of p53heterozygosity, redistributes and expands the tumor spectrum leading to the formation of angiosarcomas and increased lymphomagenesis. Importantly, we find that Pmlinactivation decreases the rate of loss of heterozygosity (LOH) in the remaining p53allele, revealing the relevancy of p53 haploinsufficiency to tumorigenesis. Our results thus lend in vivogenetic support to the importance of the crosstalk between these two critical tumor suppressor genes.

Altered microRNA (miRNA) expression may occur early in bladder cancer and may play a role in carcinogenesis and tumor behavior. We evaluated whether alterations in miRNA expression could improve disease stratification and outcome prognosis in bladder tumors and noninvasive diagnosis in urinary samples. miR-143, miR-222, and miR-452 expression levels were analyzed by quantitative RT-PCR (RT-qPCR) in paired urinary and matching tumors and in two independent prospective series of tumors and urinary specimens. Differential expression of miR-143, miR-222, and miR-452 in urine were verified by in situhybridization in matching tumors. Tumor miRNA expression by RT-qPCR correlated with tumor grade, size, and presence of carcinoma in situfor miR-222, recurrence (miR-222 and miR-143), progression (miR-222 and miR-143), disease-specific survival (miR-222), and overall survival (miR-222). Protein expression patterns of potential miRNA targets, including vascular endothelial growth factor, BCL2, v-erb-b2 erythroblastic leukemia viral oncogene (ERBB) homolog 3, and ERBB4, were evaluated by IHC in tissue arrays containing tumors for which miRNAs were assessed by RT-qPCR. Target expression correlated with expression of their predicted regulatory miRNAs, recurrence (ERBB3), progression (ERBB4), disease-specific survival (ERBB3 and ERBB4), and overall survival (ERBB3 and ERBB4). Furthermore, RT-qPCR of miR-452 (area under the curve, 0.848) and miR-222 (area under the curve, 0.718) in urine provided high accuracies for bladder cancer diagnosis. Thus, bladder tumors were characterized by changes in miRNA expression that could aid in tumor stratification and clinical outcome prognosis, and miRNAs were detected in urinary specimens for noninvasive diagnosis.

Translocation renal cell carcinoma is a newly recognized subtype of renal cell carcinoma (RCC) with chromosomal translocations involving TFE3(Xp11.2) or, less frequently, TFEB(6p21). Xp11 translocation RCC was originally described as a pediatric neoplasm representing 20 to 40 of pediatric RCCs, with a much lower frequency in the adult population. TFEBtranslocation RCC is very rare, with approximately 10 cases reported in the literature. Here, we describe the clinicopathologic features of adult translocation RCC from a single institution. Using tissue microarray, immunohistochemistry, cytogenetic examination, and fluorescence in situ hybridization, we identified 6 (∼5) cases of TFE3translocation RCC and 1 (<1) case of TFEBtranslocation RCC in 121 consecutive adult RCC cases between 2001 and 2009. Our results suggest that weak TFE3 staining of a significant proportion of RCC cases may be because of expression of the full-length TFE3 protein rather than the chimeric fusion protein resulting from chromosomal translocation.

KISS1is a metastasis suppressor gene that is lost in several malignancies, including bladder cancer. We tested the epigenetic silencing hypothesis and evaluated the biological influence of KISS1methylation on its expression and clinical relevance in bladder cancer. KISS1hypermethylation was frequent in bladder cancer cells analyzed by methylation-specific PCR and bisulfite sequencing and was associated with low gene expression, being restored in vitroby demethylating azacytidine. Hypermethylation was also frequently observed in a large series of bladder tumors (83.1%, n= 804). KISS1methylation was associated with increasing stage (P= 0.001) and tumor grade (P= 0.010). KISS1methylation was associated with low KISS1transcript expression by quantitative RT-PCR (P= 0.037). KISS1transcript expression was also associated with histopathological tumor stage (P< 0.0005). Low transcript expression alone (P= 0.003) or combined with methylation (P= 0.019) was associated with poor disease-specific survival (n= 205). KISS1transcript expression remained an independent prognosticator in multivariate analyses (P= 0.017). KISS1hypermethylation was identified in bladder cancer, providing a potential mechanistic explanation (epigenetic silencing) for the observed loss of KISS1in uroepithelial malignancies. Associations of KISS1methylation and its expression with histopathological variables and poor survival suggest the utility of incorporating KISS1measurement using paraffin-embedded material for tumor stratification and clinical outcome prognosis of patients with uroepithelial neoplasias.

The TP63gene, a member of the TP53tumor suppressor gene family, can be expressed as at least six isoforms due to alternative promoter use and alternative splicing. The lack of p63 isoform–specific antibodies has limited the analysis of the biological significance of p63. We report a novel set of well-defined antibodies to examine p63 isoforms in mouse and human urothelium during embryogenesis and tumor progression, respectively. We provide evidence that basal and intermediate urothelial cells express p63 isoforms, with the TAp63 variant the first to be detected during development, whereas umbrella cells are characterized by a p63-negative phenotype. Notably, we report that p63-null mice develop a bladder with an abnormal urothelium, constituted by a single layer of cells that express uroplakin II and low molecular weight cytokeratins, consistent with an umbrella cell phenotype. Finally, analysis of 202 human bladder carcinomas revealed a new categorization of invasive tumors into basal-like (positive for ΔNp63 and high molecular weight cytokeratins and negative for low molecular weight cytokeratins) versus luminal-like (negative for ΔNp63 and high molecular weight cytokeratins and positive for low molecular weight cytokeratins) phenotypes, with ΔNp63 expression associated with an aggressive clinical course and poor prognosis. This study highlights the relevance of p63 isoforms in both urothelial development and bladder carcinoma progression, with ΔNp63 acting as an oncogene in certain invasive bladder tumors.

The promyelocytic leukemia gene (PML) of acute promyelocytic leukemia is an established tumor suppressor gene with critical functions in growth suppression, induction of apoptosis, and cellular senescence. Interestingly, although less studied, PML seems to play a key role also in immune response to viral infection. Herein, we report that Pml-/- mice spontaneously develop an atypical invasive and lethal granulomatous lesion known as botryomycosis (BTM). In Pml-/- mice, BTM is the result of impaired function of macrophages, whereby they fail to become activated and are thus unable to clear pathogenic microorganisms. Accordingly, Pml-/- mice are resistant to lipopolysaccharide (LPS)–induced septic shock as a result of an ineffective production of cytokines and chemokines, suggesting a role for PML in the innate immune Toll-like receptor (TLR)/NF-?B prosurvival pathway. These results not only shed light on a new fundamental function of PML in innate immunity, but they also point to a proto-oncogenic role for PML in certain cellular and pathological contexts.

There has been a fundamental shift in biopharmaceutical research from a linear, systematic investigation to a more whole organism, systems-oriented analysis program. The impetus behind this change is an acknowledgment that the current novel target and drug discovery model was flawed due in part to an overly simplified view of disease biology, i.e., a unidirectional path from one gene to one protein to one mechanism of action. Fortunately, recent technological advancements in whole genome analysis, DNA, RNA, proteome sequencing and mathematical modeling have permanently altered the target discovery landscape. These improvements in high throughput knowledge-creation have facilitated the multi-dimensional interrogation of disease in the context of the whole organism. One of the recently described mechanisms for helping to bridge these investigational studies with clinical medicine has been through the introduction of an analytic modeling platform known as “systems pathology”. The derived systems-based pathology models use the patients' own clinical data and intact tissue specimens to construct a baseline phenotype for defining a clinical risk state. These biologic-quantitative models also provide a biomarker profile which can be linked to treatment and health outcomes. This paper presents the rationale and implementation of systems biology in the area of translational medicine and a practical clinical application, i.e., systems pathology. By incorporating high dimensional genome analysis and disease modeling efforts such as systems pathology, we illustrate how such advancements have helped bring systems biology to the clinic but have also served to make the medical decision and treatment algorithms a more patient-specific paradigm.

Within healthy human somatic cells, retrotransposition by long interspersed nuclear element-1 (also known as LINE-1 or L1) is thought to be held in check by a variety of mechanisms, including DNA methylation and RNAi. The expression of L1-ORF1 protein, which is rarely found in normal tissue, was assayed using antibodies with a variety of clinical cancer specimens and cancer cell lines. L1-ORF1p expression was detected in nearly all breast tumors that the authors examined, and the protein was also present in a high percentage of ileal carcinoids, bladder, and pancreatic neuroendocrine tumors, as well as in a smaller percentage of prostate and colorectal tumors. Tumors generally demonstrated cytoplasmic L1-ORF1p; however, in several breast cancers, L1-ORF1p was nuclear. Patients with breast tumors displaying nuclear L1-ORF1p had a greater incidence of both local recurrence and distal metastases and also showed poorer overall survival when compared with patients with tumors displaying cytoplasmic L1-ORF1p. These data suggest that expression of L1-ORF1p is widespread in many cancers and that redistribution from cytoplasm to nucleus could be a poor prognostic indicator during breast cancer. High expression and nuclear localization of L1-ORF1p may result in a higher rate of L1 retrotransposition, which could increase genomic instability.

The importance of adult stem cells in the development of neoplastic diseases is becoming increasingly well appreciated. We hypothesized that sarcomas of soft tissue could be categorized by their developmental/differentiation status from stem cell to mature tissue, similar to the hematological malignancies. We conducted gene expression analyses during in vitrodifferentiation of human mesenchymal stem cells into adipose tissue, as a representative mature connective tissue, and identified genes whose expression changed significantly during adipogenesis. Gene clustering and distance correlation analysis allowed the assignment of a unique time point during adipogenesis that strongly correlates to each of the four major liposarcoma subtypes. Using a novel gene expression strategy, in which liposarcomas are compared to their corresponding adipocytic maturing cells, we identified a group of genes overexpressed in liposarcomas that indicate the stage of differentiation arrest, ie, sharing a similar expression profile to adipocytic cells at a corresponding stage of differentiation, and a distinct set of genes overexpressed in liposarcomas that are not found in the corresponding stage of differentiation. We propose that the latter set is enriched for candidate transformation-associated genes. Our results indicate that a degree of developmental maturity can be quantitatively assigned to solid tumors, supporting the notion that transformation of a solid tumor stem cell can occur at distinct stages of maturation.

The Wnt signal transduction pathway coordinates myriad activities, from development and differentiation to proliferation and tumorigenesis. What is perhaps most remarkable is that Wnt signaling is able to accomplish this diverse set of activities in a cell-specific and differentiation stage-dependent manner. In this review, we will highlight the diverse effect of Wnt signaling on three types of tissue stem cells and their corresponding malignancies.